WO2015020522A1 - Composition d'homologues de crumbs et vaa recombinants et procédés pour traiter la lca-8 et la rp progressive - Google Patents

Composition d'homologues de crumbs et vaa recombinants et procédés pour traiter la lca-8 et la rp progressive Download PDF

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WO2015020522A1
WO2015020522A1 PCT/NL2014/050549 NL2014050549W WO2015020522A1 WO 2015020522 A1 WO2015020522 A1 WO 2015020522A1 NL 2014050549 W NL2014050549 W NL 2014050549W WO 2015020522 A1 WO2015020522 A1 WO 2015020522A1
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protein
modified
crb3
crb2
amino acid
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PCT/NL2014/050549
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English (en)
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Jan Wijnholds
Lucie Pierrette Françoise PELLISSIER
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Koninklijke Nederlandse Akademie Van Wetenschappen
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Priority to US14/910,302 priority Critical patent/US11246947B2/en
Priority to CA2919995A priority patent/CA2919995A1/fr
Priority to AU2014305218A priority patent/AU2014305218B2/en
Priority to EP14761426.7A priority patent/EP3030665B9/fr
Priority to JP2016533272A priority patent/JP6827320B2/ja
Priority to CN201480054880.9A priority patent/CN105980569A/zh
Publication of WO2015020522A1 publication Critical patent/WO2015020522A1/fr
Priority to IL243954A priority patent/IL243954B/en
Priority to US17/558,052 priority patent/US20220193258A1/en

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Definitions

  • This invention relates to the fields of molecular biology, virology and gene therapy.
  • the invention relates to the treatment of a retinal disorder due to mutations in Crumbs homologue- 1 (CRB1) using a gene therapy vector.
  • CRB1 Crumbs homologue- 1
  • LCA Leber's congenital amaurosis
  • LCA type 8 (LCA8) is inherited recessively. Symptoms of LCA8 patients include nystagmus, slow pupil response, retinal dysfunction, impaired vision and ultimately blindness. The eyes of these blind or severely visually impaired infants show an apparent normal fundus but lack of retinal activity as measured by electroretinography (ERG). At least some LCA8 patients show a thicker retina than normal or other LCA patients. LCA8 patients have mutations or DNA alterations in or affecting the CRBl gene locus. LCA8 patients account for 10- 15% of all LCA patients.
  • Retinitis pigmentosa is an inherited and severe group of degenerative eye disease that occurs at 1 in 3.000 people and causes severe vision impairment and often results in complete blindness. Severe recessive progressive retinitis pigmentosa occurs in young children that have mutations or DNA alterations in or affecting the Crumbs homologue-1 (CRBl) gene locus. These young children become gradually blind before their twentieth birth day. There is no clear relation between genotype (type of mutation) and phenotype (LCA or RP). RP due to mutations in the CRBl gene (RP12) account for 3-5% of all RP patients.
  • CRBl CRBl retinitis pigmentosa with para-arteriolar preservation of the RPE (PPRPE); recessive retinitis pigmentosa; recessive Leber congenital amaurosis; or dominant pigmented paravenous chorioretinal atrophy.
  • the symptoms may include Coats-like exudative vasculopathy; mutations result in a thickened retina with abnormal lamination.
  • LCA is mostly monogenic but caused by more than 20 genes, including CRBl (-10-15% of all cases), CEP290 (-20% of all cases), GUCY2D (-15% of all cases), IMPDH1 (-10% of all cases), RPE65 (-5% of all cases), and the less frequently occurring AIPLl, RPGRIP1, RDH12, MNAT1, SPATA7, LCA5, CRX, TULP1, MERTK, LRAT, RD3, OTX, CABP4, KCNJ13, IQCB1 and others (den Hollander et al., 2009). Mutations in the CRBl gene are a leading cause of LCA (10-15% of all cases). Genetic analyses showed that RP is caused by more than 50 genes.
  • CRBl Crumbs homologue-1
  • loss of CRB l results in loss of adhesion between retinal progenitor cells and newly differentiated photoreceptors and Miiller glia cells. Ultimately, the misplaced cells do not form a functional neuronal network and undergo cell death. Loss of CRB l in the developing retina also results in an increase in number of late born retinal cells (rod photoreceptors, Miiller glia cells, bipolar cells, late born sub-types of amacrine cells) and an increase in mislocalized retinal cells causing an immature appearance of the retina.
  • the methods and means have no toxicity or almost no toxicity.
  • the present invention has sought to provide a gene therapy vector to be used for the treatment of retinal disorders due to mutations in CRB1.
  • the present invention relates to a gene therapy vector for use as a medicament, wherein the gene therapy vector comprises: a) a nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein; and/or b) a nucleotide sequence encoding a modified Crumbs homologue-1 (CRB1) protein or a modified Crumbs homologue-3 (CRB3) protein, wherein the modified CRB1 protein or the modified CRB3 protein comprises a modification in the C-terminal part of the protein.
  • the modification in the C-terminal part of the protein of the modified CRB 1 or CRB3 proteins reduce the toxicity of the proteins, as can be determined in an in vivo assay.
  • the modified CRB1 protein or the modified CRB3 protein is less toxic or not toxic in an in vivo assay.
  • an in vivo assay comprises intravitreal transduction in one eye (e.g. the left eye) of a mouse retina lacking CRB2 or having reduced CRB2 protein levels as compared to a wild-type mouse retina, preferably of a Crb2 conditional knock-out (cKO) or CrblCrb2 cKO mouse, more preferably of a Crb2 F F ChxlOCre/+ cKO or Crbl "/" Crb2 F/+ ChxlOCre/+ cKO mouse, with AAV particles containing the nucleotide sequence encoding the modified CRB 1 or modified CRB3 protein and in the other eye (e.g.
  • a nucleotide sequence encoding a wild-type CRB2 protein, wild-type CRB 1 or wild-type CRB3 as a control and determination of the electroretinogram one and three months after transduction, wherein an increased percentage in the maximum a-wave and/or b-wave amplitude (in microvolts) in the electroretinogram in the modified CRB1 or modified CRB3 transduced retinas compared to the electroretinogram in the wild-type CRB1 or wild-type CRB3 transduced retinas indicates that the modified CRB1 protein or the modified CRB3 protein, with amino acid substitutions with substantial identity to the C-terminal part of the CRB2 protein, is less toxic or wherein the maximum a-wave and/or b-wave amplitude (in microvolts) in the electroretinogram is at least 60% of the difference of maximum a-wave and/or b-wave amplitude with wild type CRB2 subtracted with the maximum a-wave and/or b-wave
  • the medicament is for use in treatment or prophylaxis of a retinal disorder due to mutations in the CRBl gene. More preferably, the retinal disorder is Leber's congenital amaurosis or retinitis pigmentosa, preferably LCA8 or RP12.
  • At least one of: a) the CRB2 protein is a eumetazoan
  • CRB2 protein preferably a CRB2 protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin, more preferably the CRB2 protein is a human CRB2 protein;
  • the modified CRBl protein is a modified eumetazoan CRBl protein, preferably a modified CRBl protein of human, non- human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin, more preferably the modified CRBl protein is a modified human CRBl protein;
  • the modified CRB3 protein is a modified eumetazoan CRB3 protein, preferably a modified CRB3 protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine
  • the gene therapy vector is a recombinant parvoviral vector or a lentiviral vector, more preferably wherein the vector is a recombinant adeno-associated virus (rAAV) vector. More preferably, the gene therapy vector is a recombinant adeno-associated virus vector selected from the group consisting of recombinant adeno-associated virus serotype 1 (rAAVl), recombinant adeno- associated virus serotype 2 (rAAV2), recombinant adeno-associated virus serotype 3 (rAAV3), recombinant adenoassociated virus serotype 4 (rAAV4), recombinant adeno- associated virus serotype 5 (rAAV5), recombinant adeno-associated virus serotype 6 (rAAV6), recombinant adeno-associated virus serotype 7 (rAAV7), recombinant adeno-
  • the CRB2 protein comprises or consists of an amino acid sequence that has at least 80% sequence identity with the amino acid sequences of any one of SEQ ID NO: 40 - 63, 65 - 83, more preferably any one of SEQ ID NO: 40 - 42 and wherein preferably the CRB2 protein is functionally active as measured by electroretinography.
  • the nucleotide sequence encoding CRB2, modified CRB l or modified CRB3 is operably linked to expression control elements comprising a promoter that produces sufficient expression of CRB2, modified CRBl or modified CRB3 protein, respectively, to obtain a therapeutic effect
  • the promoter preferably is selected from the group consisting of: truncated CMV promoter, CMV promoter, truncated human RLBPl promoter, human photoreceptor specific rhodopsin kinase promoter, and human rod photoreceptor specific rhodopsin promoter, wherein preferably the promoter is selected from the group consisting of: CMV promoter according to SEQ ID NO: 121, truncated human RLBPl promoter according to SEQ ID NO: 122, human photoreceptor specific rhodopsin kinase promoter according to SEQ ID NO: 123, human rod photoreceptor specific rhodopsin promoter according to S
  • the modification in the C-terminal part of the amino acid sequence of the modified CRBl or modified CRB3 protein is selected from the group consisting of: i) the PDZ binding domain of CRBl or CRB3 is replaced by amino acid residues 1282 - 1285 of SEQ ID NO:40; ii) the FERM binding domain of CRB l or CRB3 is replaced by amino acid residues 1251 - 1264 of SEQ ID NO:40; iii) the transmembrane domain of CRB l or CRB3 is replaced by amino acid residues 1225 - 1247 of SEQ ID NO:40; iv) the 16 C-terminal amino acid residues of CRB l or CRB3 are replaced by amino acid residues 1270 - 1285 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1270 - 1285 of SEQ ID NO:40; v) an amino acid sequence of CRBl or CRB3 consisting of the FERM binding domain
  • the present invention relates to a nucleic acid construct comprising at least one of : a) a nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein and at least one parvoviral inverted terminal repeat (ITR) sequence, wherein preferably the nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein is operably linked to expression control elements comprising a promoter that is capable of sufficient CRB2 protein expression to obtain a therapeutic effect; and b) a nucleotide sequence encoding a Crumbs homologue-1 (modified CRBl) protein or a Crumbs homologue-3 (modified CRB3) protein, and at least one ITR sequence, wherein preferably the nucleotide sequence encoding a modified CRBl protein or a modified CRB3 protein is operably linked to expression control elements comprising a promoter that is capable of sufficient modified CRBl or modified CRB3 protein expression to obtain a therapeutic effect and wherein the
  • rAAV comprising the nucleotide sequence encoding the modified CRBl or modified CRB3 protein
  • rAAV intravitreal transduction in the other eye of the mouse with rAAV comprising a nucleotide sequence encoding a wild-type CRB2 protein, wild-type CRBl or wild-type CRB3 as a control;
  • a maximum a-wave and/or b-wave amplitude in the electroretinogram of at least 60% of the difference of maximum a-wave and/or b-wave amplitude with wild type CRB2 subtracted with the maximum a- wave and/or b-wave amplitude with modified CRBl or modified CRB3 protein indicates that the modified CRBl protein or the modified CRB3 protein is not toxic.
  • the modification in the C-terminal part of the amino acid sequence of the modified CRBl or modified CRB3 protein is selected from the group consisting of: i) the PDZ binding domain of CRBl or CRB3 is replaced by amino acid residues 1282 - 1285 of SEQ ID NO:40; ii) the FERM binding domain of CRB l or CRB3 is replaced by amino acid residues 1251 - 1264 of SEQ ID NO:40; iii) the transmembrane domain of CRB l or CRB3 is replaced by amino acid residues 1225 - 1247 of SEQ ID NO:40; iv) the 16 C-terminal amino acid residues of CRBl or CRB3 are replaced by amino acid residues 1270 - 1285 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1270 - 1285 of SEQ ID NO:40; v) an amino acid sequence of CRBl or CRB3 consisting of the FERM binding domain of
  • the present invention relates to a nucleic acid construct according to the invention, wherein preferably, the virion is an AAV virion.
  • the present invention relates to a host cell comprising the nucleic acid construct according to the invention.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a gene therapy vector according to the invention, a nucleic acid construct according to the invention, or a virion according to the invention and a pharmaceutically acceptable excipient.
  • the present invention relates to a kit comprising: (a) a gene therapy vector in accordance with the invention, a nucleic acid construct according to the invention, a virion according to the invention, or a pharmaceutical composition according to claim the invention; and, (b) optionally, instructions for using the gene therapy vector or pharmaceutical composition according to (a) in the prevention, treatment, or amelioration of one or more symptoms of a retinal disorder due to mutations in CRB1 gene.
  • insect cell refers to an insect cell which allows for replication of a recombinant parvoviral (rAAV) vector and which can be maintained in culture.
  • the cell line used can be from Spodoptera frugiperda, Drosophila cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines.
  • Preferred insect cells or cell lines are cells from the insect species which are susceptible to baculovirus infection, including e.g.
  • operably linked refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. The phrase "under control of is used interchangeably herein.
  • “Expression control sequence” refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked.
  • An expression control sequence is "operably linked" to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence.
  • an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons.
  • the term "expression control sequence” is intended to include, at a minimum, a sequence whose presence are designed to influence expression, and can also include additional advantageous components.
  • leader sequences and fusion partner sequences are expression control sequences.
  • the term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3'-end of a mRNA, sequences referred to as polyA sequences. It also can be designed to enhance mRNA stability.
  • pA polyadenylation sequences
  • Expression control sequences which affect the transcription and translation stability e.g., promoters, as well as sequences which effect the translation, e.g., Kozak sequences, are known in insect cells.
  • Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved.
  • promoter or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.
  • a "tissue specific” promoter is only active in specific types of tissues or cells.
  • substantially identical means that two peptide or two nucleotide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein.
  • RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
  • Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA or the open-source software Emboss for Windows (current version 2.7.1-07).
  • percent similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc.
  • nucleic acid construct is intended to mean a nucleic acid molecule (typically comprised of DNA) operably linked to expression control elements, such as for example a promoter that is capable of expression of the nucleic acid molecule.
  • gene therapy vector is generally intended to mean a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment.
  • a virus is an exemplary gene therapy vector.
  • vector refers to a genetic construct that is composed of genetic material (i.e., nucleic acids).
  • Vectors may include one or more genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in a suitable expression cell.
  • the vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.
  • a gene therapy vector disclosed herein may optionally be comprised within an infectious viral particle.
  • the terms "viral particle” and “virion” are used interchangeably herein.
  • the present invention also encompasses virions as well as host cells that comprise a nucleic acid construct or a gene therapy vector of the invention.
  • protein As used herein, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably, and include molecules that include at least one amide bond linking two or more amino acid residues together.
  • a peptide is a relatively short (e.g., from 2 to about 100 amino acid residues in length) molecule, while a protein or a polypeptide is a relatively longer polymer (e.g., 100 or more residues in length).
  • peptide, polypeptide, and protein are used interchangeably.
  • the term "subject” refers to any subject that can serve as a recipient for a gene therapy vector, a pharmaceutical composition, or a virion of the present invention.
  • the subject will be a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being).
  • a "patient” refers to any animal host, including but not limited to, human and non-human primates, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.
  • an effective amount would be understood by those of ordinary skill in the art to provide a therapeutic, prophylactic, or otherwise beneficial effect to a recipient subject.
  • isolated refers to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state.
  • isolated polynucleotides in accordance with the invention preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.
  • amino acid substitutions in the same functional class relate to so-called “conservative” amino acid substitutions, as will be clear to the skilled person.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having small side chains is alanine, serine, threonine, methionine and glycine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having acidic side chains is as
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg, gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr or gly; Thr to ser or val; Trp to tyr; Tyr to tip or phe; and, Val to ile or leu.
  • Detailed description of the invention are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu
  • the present invention relates to the treatment of a retinal disorder or a disorder associated with cellular changes in the retina, in particular changes due to one or more mutations in CRBl . More specifically, the present invention relates to the treatment of Leber's congenital amaurosis (LCA), in particular Leber's congenital amaurosis-8 (LCA8), and to the treatment of progressive retinitis pigmentosa (RP), in particular progressive retinitis pigmentosa 12 (RP12), or alternatively said early onset RP12.
  • LCA Leber's congenital amaurosis
  • RP progressive retinitis pigmentosa
  • RP12 progressive retinitis pigmentosa 12
  • the invention provides a method for at least in part decreasing loss of retinal activity and structural integrity in an animal, wherein the loss of retinal activity and structural integrity comprising at least in part loss of Crumbs homologue (CRB) function in said animal.
  • said decreasing of loss of retinal activity and structural integrity is accomplished via a recombinant adeno-associated viral (rAAV) expression vector expressing a first nucleic acid segment encoding a first therapeutic gene product that express a biologically-functional Crumbs homologue (CRB) peptide, polypeptide, or protein for use in one or more investigative, diagnostic and/or therapeutic regimens, including for example, the treatment of one or more disorders or diseases of the mammalian eye, and in particular, for treating congenital retinal blindness including, retinal dystrophy such as Leber's congenital amaurosis type 8 (LCA8) and retinitis pigmentosa (RP) due to lack of sufficient biological Crumbs homologue (CRB) function, in humans. It is preferred that the treatment is not or almost not toxic.
  • rAAV adeno-associated viral
  • the present invention relates to a gene therapy vector for use as a medicament, wherein the gene therapy vector comprises: a) a nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein; and/or b) a nucleotide sequence encoding a modified Crumbs homologue-1 (CRBl) protein or a modified Crumbs homologue-3 (CRB3) protein, wherein the modified CRBl protein or the modified CRB3 protein comprises a modification in the C-terminal part of the protein.
  • CB2 Crumbs homologue-2
  • CB3 modified Crumbs homologue-3
  • the C-terminal part of the modified CRBl protein or the modified CRB3 protein has substantial identity with the C-terminal part of the CRB2 protein, preferably at least 85, 90, 93, 95, 97, 98 or 99% identity with the C-terminal part of the CRB2 protein, as is further defined below.
  • the modification in the C-terminal part of the protein of the modified CRBl CRB3 proteins reduces the toxicity of the proteins, as can be determined in an in vivo assay.
  • the gene therapy vector comprising the sequence encoding for modified CRBl or modified CRB3 protein is less toxic than the wild-type CRBl or wild-type CRB3 protein, more preferably the gene therapy vector comprising the sequence encoding for modified CRB l or modified CRB3 protein is almost not or not toxic in an in vivo assay.
  • the assay comprises intravitreal transduction of mouse retinas with reduced levels of or lacking CRB2, preferably mouse retinas lacking CRBl and having reduced levels of CRB2, in one eye with AAV particles containing the nucleotide sequence encoding the modified CRBl or modified CRB3 protein and in the other eye with a nucleotide sequence encoding a wild-type CRB2 protein, wild-type CRBl or wild-type CRB3 as a control and determination of the electroretinogram one and three months after transduction, wherein an increased percentage in the maximum a-wave and/or b-wave amplitude (in microvolts) in the electroretinogram in the modified CRBl or modified CRB3 transduced retinas compared to the electroretinogram in the wild-type CRBl or wild- type CRB3 transduced retinas indicates that the modified CRB 1 protein or the modified CRB3 protein is less toxic or wherein the maximum a-wave and/or b-wave amplitude (in microvolts) in
  • a mouse e.g. the left eye
  • intravitreal transduction in one eye of a mouse with reduced levels of or lacking CRB2, preferably of a mouse lacking CRB l and having reduced levels of CRB2, more preferably of a Crb2 conditional knock-out (cKO) or CrblCrb2 F/+ cKO mouse, more preferably of a Crb2 F F ChxlOCre/+ cKO or Crbl 7" Crb2 F/+ ChxlOCre/+ cKO mouse, with 1 ⁇ _, of 5.10 9 genome copies genome copies genome copies of recombinant adeno-associated virus (rAAV) comprising the nucleotide sequence encoding the modified CRBl or modified CRB3 protein, wherein preferably the rAAV is ShHIOY and wherein the nucleotide sequence encoding the modified CRBl or the modified CRB3 is operably linked to a minimal CMV or a CMV promoter, respectively;
  • rAAV
  • rAAV intravitreal transduction in the other eye of the mouse (e.g. the right eye) with 1 ⁇ _, of 5.10 9 genome copies genome copies of rAAV comprising a nucleotide sequence encoding a wild-type CRB2 protein, wild-type CRBl protein or wild-type CRB3 protein as a control, wherein preferably the rAAV is ShHIOY and wherein the nucleotide sequence encoding the wild-type CRB2, CRBl or CRB3 protein is operably linked to a CMV, minimalCMV, or a CMV promoter, respectively; and, iii) making an electroretinogram one and three months after transduction, wherein an increased percentage in the maximum a- wave and/or b-wave amplitude in the electroretinogram in the modified CRBl or modified CRB3 transduced retinas compared to the electroretinogram in the wild-type CRBl or wild-type CRB3 transduced retinas indicates that the modified CRB l
  • a maximum a-wave and/or b-wave amplitude in the electroretinogram of at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the difference of maximum a-wave and/or b-wave amplitude with wild type CRB2 subtracted with the maximum a-wave and/or b-wave amplitude with modified CRBl or modified CRB3 protein indicates that the modified CRBl protein or the modified CRB3 protein is not toxic.
  • the gene therapy vector comprising the sequence encoding for modified CRBl or modified CRB3 protein is almost not or not toxic in an in vitro assay.
  • the in vitro assay comprises transfection of human-derived retinal pigment epithelial cells with the nucleotide sequence encoding the modified CRBl or modified CRB3 protein or with a nucleotide sequence encoding CRBl protein, CRB2 protein or CRB3 protein as a control and determination of cell viability 72 hours after transfection.
  • the CRBl, CRB2, and CRB3 control proteins are wild-type CRB l, CRB2, and CRB3 proteins.
  • the modified CRBl protein or the modified CRB3 protein having a higher percentage of viable cells than wild-type CRBl protein or wild-type CRB3 protein indicates that the modified CRBl protein or the modified CRB3 protein is less toxic than the wild-type CRBl protein or the wild- type CRB3 protein.
  • a percentage of viable cells of at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the percentage of viable cells of control CRB2 protein indicates that the modified CRBl protein or the modified CRB3 protein is not toxic.
  • the in vitro assay preferably comprises transfection of human- derived retinal pigment epithelial cells with the nucleotide sequence encoding the modified CRBl or modified CRB3 protein together with a nucleotide sequence encoding green fluorescent protein as a transfection control.
  • Human-derived retinal pigment epithelial cells transfected with CRB2 were used as a control. Viability of the cells was determined 72 hours after transfection.
  • the modified CRBl protein or the modified CRB3 protein is considered non-toxic if the amount of viable cells after treatment of the cells with the nucleotide sequence encoding any of these proteins is at least 80, 90 or 95% of the amount of viable cells in the CRB2 treated control cells.
  • the amount of modified CRBl protein or modified CRB3 protein expressed by the cells is determined, such as for example in an ELISA or a Western Blot. In the example a method for Western blot is provided. If the cells treated with the nucleotide sequence encoding the modified CRB l protein or the modified CRB3 protein show more protein expression than the wild-type CRBl or wild-type CRB3 control, then the modified CRB l protein or the modified CRB3 protein is considered less toxic.
  • the modified CRBl protein or the modified CRB3 protein shows at least 60, 65, 70, 75, 80, 85, 90, 95 or 100% protein expression of the CRB2 protein expression in the CRB2 control cells (preferably normalized to a housekeeping protein, such as for example actin protein levels), then the modified CRBl protein or the modified CRB3 protein is considered non-toxic.
  • the CRB construct is not toxic.
  • the percentage of viable cells after transfection with the nucleotide sequence encoding modified CRBl protein or modified CRB3 protein is less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60%, of the percentage of viable cells after transfection with a nucleotide sequence encoding CRB2 protein, then the construct is said to be toxic.
  • the human-derived retinal pigment epithelial cells are ARPE19 cells, preferably obtained from ATCC CRL-2302.
  • the gene therapy vector comprises a nucleotide sequence encoding a CRB2 protein.
  • the full length CRBl (for example, SEQ ID NO: 1 - 2) cannot usually be used in adeno-associated virus because of its size, although recently it has been shown that it is possible to express full length CRBl using AAV9 when using a small promoter, such as a truncated CMV (minimal CMV; preferably SEQ ID NO: 133) or hGRKl promoter (Pellissier et al. (2014) Mol Ther Methods&Clinical Dev7: 14009).
  • the size of normal full length CRBl cDNA is about 4.22 kb.
  • a vector comprising this sequence and also comprising other expression elements such as a CMV promoter, inverted terminal repeats and 5' untranslated region would approximately be 5.2 kb.
  • the gene therapy vector of the present invention does not comprise a nucleotide sequence encoding naturally occurring CRBl and/or CRB3 protein and does not comprise a nucleotide sequence encoding a naturally occurring short variant of CRBl (lacking EGF domains as compared to full-length CRBl).
  • these sequences are provided in the sequence listing (e.g. SEQ ID NO: 1 - 39 and 64 for CRB l and SEQ ID NO: 84 - 120 for CRB3).
  • the present inventors found that CRB2 did not result in a significant toxic effect as seen with the naturally occurring short variant of CRBl and with CRB3.
  • mice lacking CRB l mice lacking CRB2
  • mice lacking CRB2 with reduced levels of CRBl mice lacking both CRB1 and CRB2
  • CRB1 and CRB2 the functions of Crumbs homologue (CRB) proteins are exchangeable e.g. the human CRB1 protein can rescue partially the phenotype in fruit flies lacking Crumbs (Crb) protein (den Hollander et al., 2001), and the zebrafish CRB2B protein can rescue the phenotype in zebrafish lacking CRB2A protein (Omori & Malicki, 2006).
  • Crb Crumbs homologue
  • Other advantages of endogenously or exogenously increasing levels of human CRB2 protein are the following:
  • human CRB2 cDNA is small, about 3.9 kb, resulting in an expression cassette comprising the CRB2 cDNA and expression elements of typically only about 4.9 kb. It was found that expression of human CRB2 using a parvoviral vector can be obtained in the retina.
  • Native CRB2 is present in the retina of mice both in photoreceptor cells and in Miiller glia cells, and possibly also in retinal pigment epithelial cells. Also in other species native CRB2 is present and functional in photoreceptor cells. However, in humans native CRB2 only is present in Miiller glia cells, more specifically at the subapical region adjacent to adherens junctions at the outer limiting membrane in Miiller glia cells, but not in photoreceptor cells. It is acknowledged that the situation between mice and man differs (figures 1 and 2). Several mouse models have been developed and experiments were performed in several mouse models. In the Examples it has been illustrated that conditional knockout mice have been developed that have a similar phenotype as presented in humans suffering from RP12.
  • C The immune system in humans who are deficient in CRB1 may recognize recombinant CRB1 as a non-self protein and possibly an immuno reaction against the recombinant CRB1 is incurred.
  • CRB2 is recognized as a self protein since it is already expressed and immune-tolerated in the retinas and epithelia of other organs of these patients and will not result in an immune response.
  • the cause of the toxicity of full length and short CRB1 as referred to above is yet unknown and may be on the level of DNA, RNA or protein. Without wishing to be bound by any theory, it is for example possible that overexpression of short CRB1 protein scavenges essential proteins or that the RNA transcript of short CRB1 cDNA results in a disbalance in microRNAs.
  • toxicity of CRB proteins is tested using human- derived retinal pigment epithelial cells as indicated in Example 3.
  • the assay is performed as follows.
  • the in vitro assay comprises transfection of human-derived retinal pigment epithelial cells with the nucleotide sequence encoding the (modified) CRB1 or (modified) CRB3 protein together with a nucleotide sequence encoding green fluorescent protein as a transfection control.
  • ARPE19 cells (ATCC CRL-2302) are transfected with one of the different CRB constructs (e.g.
  • CRB2 construct preferably having the CRB2 sequence as shown in SEQ ID NO: 40
  • a wild-type CRB1 construct preferably having the CRB1 sequence as shown in SEQ ID NO: l or 2 or in case of wild-type short CRB1 of SEQ ID NO:3
  • a wild-type CRB3 construct preferably having the CRB3 sequence as shown in SEQ ID NO:84
  • Each CRB construct or control is tested in a separate petridish in duplo.
  • the CRB constructs are used in equimolar amounts and a total amount of 20 ⁇ g of DNA is added per petridish.
  • CRB constructs are made as described in Example 2.1.
  • CRB constructs are made by chemical synthesis and subcloned into pUC57. These constructs comprise AAV2 ITRs (SEQ ID NO: 131 and 132), CMV promoter (SEQ ID NO: 121), CRB cDNA to be tested (e.g. SEQ ID NO:40 or other CRB sequence, Intron 5 (SEQ ID NO: 128), and synthetic pA (SEQ ID NO: 130).
  • AAV2 ITRs SEQ ID NO: 131 and 132
  • CMV promoter SEQ ID NO: 121
  • CRB cDNA to be tested e.g. SEQ ID NO:40 or other CRB sequence, Intron 5 (SEQ ID NO: 128), and synthetic pA (SEQ ID NO: 130).
  • the GFP construct is used as internal transfection control and to provide a fixed amount of DNA of 20 ⁇ g to the cells. For example, 18 ⁇ g of CRB construct plus 2 ⁇ g of GFP construct is used. In this way, a series of equimolar plasmid concentrations can be tested while adding the same amount of DNA, such as for example 2, 4, 8 or 16 ⁇ g of CRB construct, plus 18, 16, 12 or 4 ⁇ g of GFP construct, respectively.
  • ARPE19 cells are plated in duplicate at 30% of confluence in a 10 cm petridish in DMEM supplemented with 10% Fetal Bovine Serine and penicillin/streptomycin. After refreshing the medium 2 hours before transfection, the transfection mix is prepared with 20 ⁇ g of DNA in 500 ⁇ of 0.25M CaCl 2 and TE (10 mM Tris, 1 mM EDTA pH 8) buffer per dish. While constantly vortexing, 500 ⁇ of 2x HBS (281 mM NaCl, 100 mM Hepes, 1.5 mM Na 2 HP0 4 , pH 7.12) are added drop wise to the transfection mixture and the complete mix is directly added to the cells for overnight incubation.
  • 2x HBS 281 mM NaCl, 100 mM Hepes, 1.5 mM Na 2 HP0 4 , pH 7.12
  • the medium is refreshed in the following morning (using DMEM supplemented with 10% Fetal Bovine Serine and penicillin/streptomycin).
  • DMEM fetal calf serum
  • the counter determines the number of cells and via Trypan Blue staining discriminates between viable and non-viable cells. Trypan Blue staining was performed using the Standard protocol by Life Technologies as further set out in Example 3. It has been described above how it can be determined using the percentage of viability whether a construct employed in this assay is toxic or not toxic.
  • CRB protein expression can be determined instead of or in addition to the percentage of viable cells.
  • CRB protein expression can be determined using any protein assay, such as for example Western Blotting as outlined in Example 3 or ELISA. Shortly, proteins from the cell lysates are separated by SDS-page electrophoresis. After transfer to nitrocellulose membrane, the nitrocellulose membrane is immunostained for CRB, GFP and Actin proteins and analyzed by Odyssey Infrared Imaging System (LI-COR; Westburg BV, Leusden, the Netherlands). Actin proteins were used for normalization of the obtained data. It has been described above how it can be determined whether a construct employed in this assay is toxic or not toxic.
  • LI-COR Odyssey Infrared Imaging System
  • toxicity of CRB proteins is tested using electroretinography on mouse retinas as indicated in Example 6 and as described for the in vivo test in more detail above.
  • the gene therapy vector according to the present invention is for use in treatment or prophylaxis of a retinal disorder due to mutations in the CRB1 gene.
  • the invention relates to the use of a gene therapy vector according to the present invention for the manufacture of a medicament in the treatment of a retinal disorder due to mutations in CRB1 gene.
  • such mutations in the CRB1 gene result in loss of CRB1 functional protein, as can be determined using electroretinography (ERG), multi-focal ERG, optical coherence tomography (OCT), microperimetry, visual evoked potention (VEP) test, functional Magnetic Resonance Imaging test, or behaviour maze-test (see for example: Bainbridge et al. (2008) N Engl J Med. May 22;358(21):2231-9; Annear et al. (2011) Gene Ther. Jan; 18(l):53-61; Maguire et al. (2008) N Engl J Med. May 22;358(21):2240-8; Testa et al. (2013) Ophthalmology.
  • the retinal disorder is Leber's congenital amaurosis or retinitis pigmentosa, more preferably LCA8 or RP12.
  • Retinitis pigmentosa is an autosomal recessive or dominant group of diseases that represent progressive or late severe forms of inherited retinal dystrophies affecting initially rod photoreceptors and subsequently cone photoreceptors.
  • the RP12 gene caused RP with preserved para-arteriolar retinal pigment epithelium (PPRPE) (Heckenlively et al., 1982).
  • LCA Leber congenital amaurosis
  • CRB1 Crumbs homologue-1
  • CRBl plays a role in the formation and maintenance of adhesion between photoreceptors and Miiller glia cells. Without CRBl protein adhesion between these cells is weakened leading to loss of normal retinal lamination. Without CRBl protein the subapical CRBl /PAL S1/MUPP1 and CRBl /PAL SI /PAT J protein complexes, required for maintaining cellular polarization and maintenance of adhesion between photoreceptors and Miiller glia cells, are destabilized.
  • CRB1 gene mutations in the CRB1 gene reduce or abolish the ability of CRB 1 protein to maintain the subapical CRB l/PALSl/MUPPl and CRB1/PALS1/PATJ protein complexes and to maintain the adhesion between photoreceptors and Miiller glia cells, as in RP with mutations in the CRB1 gene or LCA8. It is unclear why some people with CRB1 gene mutations have severe, early visual impairment associated with Leber congenital amaurosis, and other people experience more gradual vision loss and other eye problems associated with retinitis pigmentosa. Other genetic factors (such as CRB2; Alves et al, 2013) may modify the effects of CRB1 gene mutations to influence the severity of these conditions.
  • LCA8 The first report of LCA was published in 1869 by Theodor Leber. Currently, at least twenty genes have been reported to cause LCA. Mutations in CRB1 account for -15% of all cases of LCA making it one of the leading causes of LCA. Diagnosis of LCA8 is typically made within the first few months of life in an infant with severely impaired vision or total blindness, a flat electroretinogram (ERG) and involuntary eye movements (nystagmus) (Hufnagel et al, 2013).
  • ERP electroretinogram
  • nystagmus involuntary eye movements
  • Loss of normal retinal structure in LCA8 is unlike other forms of the disease which exhibit marked retinal thinning that generally worsens with age (Pasadhika et al, 2009) or exhibit preserved retinal structure with loss of retinal activity as is the case for LCA1 due to mutations in the Gucy2d gene.
  • SDOCT spectral-domain optical coherence tomography
  • Retinitis pigmentosa is the leading cause of inherited retinal degeneration-associated blindness.
  • Retinitis pigmentosa is a disease condition that was first identified and named by Dr. Donders in 1857.
  • Retinitis pigmentosa is a group of related conditions that are inherited, progressive and clinically distinctive and share a similar feature of dystrophy or damage to the photoreceptors of the retina and of the pigment epithelium underneath the photoreceptors.
  • RP Retinitis pigmentosa
  • CRB 1 function in humans leads to progressive RP12 or LCA8, though loss of CRB1 function in mice leads to relative mild retinal disorganization and degeneration. It is unclear why some people with CRB1 gene mutations have severe, early visual impairment associated with Leber congenital amaurosis, and other people experience more gradual but progressive early onset vision loss and other eye problems associated with retinitis pigmentosa. It is also unclear why mice lacking CRB1 show a relative mild phenotype compared to humans lacking CRB1. Other genetic factors (such as CRB2; Alves et al, 2013) may modify the effects of Crbl gene mutations to influence the severity of these conditions.
  • mice lacking CRB2 in the retina show a phenotype mimicking progressive RP detected in human patients lacking CRB 1 (Alves et al, 2013), and mice lacking CRB2 and CRB1 mimic LCA8 detected in human patients lacking CRB1 (Pellissier et al., PLoS Genet. 2013 Dec;9(12):el003976).
  • Other factors involved are light exposure; exposure to moderate levels of white light significantly increased the level of retinal disorganization and degeneration in mice lacking CRB1 (van de Pavert et al, 2004; van de Pavert et al., 2007a; van de Pavert et al., 2007b).
  • mice and humans may differ because of different localization of CRB1 and CRB2 proteins.
  • immuno electron microscopy showed that CRB1 localizes in the apical villi of Miiller glia cells at the subapical region (SAR) adjacent to adherens junctions (AJ) at the outer limiting membrane (OLM).
  • CRB2 localizes at two regions: the inner segments of photoreceptors at the subapical region (SAR) adjacent to adherens junctions (AJs) at the outer limiting membrane (OLM), as well as at the apical villi of Miiller glia cells at the subapical region (SAR) adjacent to adherens junctions (AJs) at the outer limiting membrane (OLM) (van Rossum et al, 2006).
  • Loss of CRB l in the mouse retina therefore leaves functional CRB2 protein in photoreceptors and Miiller glia cells, resulting in a mild phenotype.
  • CRB2 localizes in the apical villi of Miiller glia cells at the subapical region (SAR) adjacent to adherens junctions (AJ) at the outer limiting membrane (OLM).
  • CRB l localizes at two regions: the inner segments of photoreceptors at the subapical region (SAR) adjacent to adherens junctions (AJs) at the outer limiting membrane (OLM), as well as at the apical villi of Miiller glia cells at the subapical region (SAR) adjacent to adherens junctions (AJs) at the outer limiting membrane (OLM) (Pellissier et al, Hum Mol Genet. 2014 Jul 15;23(14):3759-71). Loss of CRBl in the human retina therefore leaves functional CRB2 protein at the SAR in Miiller glia cells but not in photoreceptors, resulting in a severe phenotype.
  • the Crb2 conditional knock-out mouse (Crb2 cKO) lacking CRB2 in all retinal cells except the retinal pigment epithelium (e.g. the Crb2 flox/flox ChxlOCre) (Alves et al., 2013).
  • the Crb2 conditional knock-out mouse lacking CRB2 in photoreceptors (e.g. the Crb2 flox/flox CrxCre) (Alves et al, Hum Mol Genet. 2014 Jul 1 ;23(13):3384-401).
  • the Crb2 conditional knock-out mouse lacking CRB2 in Muller glia cells (e.g.
  • the homozygote Crbl heterozygote Crb2 conditional knock-out mouse lacking CRB1 in all retinal cells and having reduced expression of CRB2 in photoreceptors (e.g. Crbl "/" Crb2 flox/+ CrxCre).
  • the homozygote Crbl heterozygote Crb2 conditional knock-out mouse lacking CRB1 in all retinal cells and having reduced expression of CRB2 in Muller glia cells (e.g. Crbl "/" Crb2 flox/+ PdgfraCre).
  • the homozygote Crbl homozygote Crb2 conditional knock-out mouse lacking CRB1 in all retinal cells and CRB2 in all retinal cells except the retinal pigment epithelium (e.g.
  • CRB1 in all retinal cells and CRB2 in photoreceptors e.g. Crbl "/" Crb2 flox/flox CrxlOCre.
  • the homozygote Crbl homozygote Crb2 conditional knock-out mouse lacking CRB1 in all retinal cells and CRB2 in Muller glia cells (e.g. Crbl 7" Crb2 flox/flox pdgfraCre)
  • the Crb2 conditional knock-out mouse lacking CRB2 in all retinal cells except the retinal pigment epithelium (e.g. the Crb2 flox/flox ChxlOCre) (Alves et al, 2013) was used to evaluate gene replacement therapy.
  • the Crb2 flox/flox ChxlOCre exhibits progressive retinal degeneration and scotopic (rod-mediated) and photopic (cone- mediated) loss of retina function as measured by ERG from 1 to 6 months of age (Alves et al, 2013).
  • the mouse is blind at 12-18 months of age.
  • AAV-mediated transfer of CRB2 to Crb2 cKO retina restored vision to these animals as evidenced by ERG.
  • fMRI functional magnetic resonance imaging
  • a gene therapy vector of the present invention is used in a combination therapy, for example in combination with a) addition of protective, nurturing or growth factors such as e.g. GD F or CT F, b) addition of drugs that normalize the intraocular pressure in eyes such as e.g. eye drops containing prostaglandin analogs, beta blockers, alpha agonists, and/or carbonic anhydrase inhibitors, c) addition of drugs or tools that decrease the light-sensitivity of eyes such as e.g. prosthetic contact lenses, d) addition of drugs that normalize the retinoid cyclus in the retina such as e.g. retinoids, e) addition of drugs that increase the strength of adherens junctions at the retinal outer limiting membrane such as e.g. magnesium and calcium salts.
  • protective, nurturing or growth factors such as e.g. GD F or CT F
  • drugs that normalize the intraocular pressure in eyes such as e.g. eye drops containing prostaglandin analogs, beta
  • the gene therapy vector of the present invention is applied only once to a subject suffering from the retinal disorder due to mutations in CRBl gene.
  • Re-application of the same or a similar vector for example with the same or another capsid, is expected to become advantageous with signs of decreased vision in the dark.
  • the same or a similar vector may be re-applied, because injection of the AAV vector subretinally provokes low immune response.
  • intravitreal injection has been demonstrated to result in an immune response, but in such cases another suitable vector may be used (Li et al. [2008] Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential. Mol Vis. 14: 1780-1789). It is envisioned that maze experiments but preferentially fMRI experiments with visual tasks in dimmed light, by testing the visual cortex, will be most instrumental in determining the time point at which a re-application becomes advantageous.
  • the subject suffering from the retinal disorder due to mutations in CRBl gene and to be treated using a gene therapy vector, a nucleic acid construct, a virion, a host cell or a pharmaceutically composition according to the present invention is a mammal, such as e.g. a human, a non-human primate, a mouse, a dog, a cat, a pork, a chicken, a monkey, a cow, a sheep, a rabbit. Most preferably, the subject is a human.
  • the CRB2 protein is a eumetazoan CRB2 protein, preferably a CRB2 protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin, more preferably the CRB2 protein is a human CRB2 protein;
  • the modified CRBl protein is a modified eumetazoan CRBl protein, preferably a modified CRBl protein of human, non-human primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, or lupine origin, more preferably the modified CRBl protein is a modified human CRBl protein; and/or c) the modified CRB3 protein is a modified eumetazoan CRB3 protein, preferably a modified CRB3 protein of human, non-human primate, murine,
  • the gene therapy vector is a recombinant parvoviral vector or a lentiviral vector.
  • Viruses of the Parvoviridae family are small DNA animal viruses.
  • the family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects.
  • Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus.
  • members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture.
  • the genus Dependovirus includes adeno-associated virus (AAV), which normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses).
  • AAV adeno-associated virus
  • AAV vectors constitute a single-stranded DNA with an outer icosahedral coat of structural protein having a diameter of 18 to 26 nm, typically about 25 nm. Further information on parvoviruses and other members of the Parvoviridae is described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996). For convenience the present invention is further exemplified and described herein by reference to AAV. It is however understood that the invention is not limited to AAV but may equally be applied to other parvoviruses.
  • AAV chimeric viruses comprising chimeric capsid proteins and/or AAV hybrid viruses (or pseudotyped viruses) that also have a similar size as found for the wild-type parvoviruses (18 - 26 nm diameter).
  • a description and some examples are given in WO0028004.
  • AAV chimeric and/or hybrid viruses are for example AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/5.2, AAV2/6, AAV2/7, AAV2/8 and AAV2/9.
  • the AAV genome consists of rep genes encoding proteins required for replication of the virus and cap genes encoding the viral structural proteins.
  • One or more of the rep genes which are required for replication e.g. rep 40, rep 52, rep 68 and/or rep 78
  • the cap genes which are required for the capsid structure e.g. VP-1, VP-2 and/or VP-3
  • the ITR regions which are still present at the 5' and 3' ends are needed, as cis-active elements, for packaging the transgene into infectious, recombinant AAV particles and for the replication of the DNA of the recombinant AAV genome (Kotin, R.
  • a "recombinant parvoviral or AAV vector” refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest or “transgenes” that are flanked by parvoviral or AAV inverted terminal repeat sequences (ITRs).
  • ITRs parvoviral or AAV inverted terminal repeat sequences
  • Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect or mammalian host cell that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins).
  • AAV Rep and Cap proteins i.e., AAV Rep and Cap proteins
  • the rAAV vector in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), then the rAAV vector is typically referred to as a "pro-vector" which can be "rescued” by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.
  • the gene therapy vector of the present invention is a rAAV vector.
  • the rAAV is selected from the group consisting of recombinant adeno-associated virus serotype 1 (rAAVl), recombinant adeno-associated virus serotype 2 (rAAV2), recombinant adeno- associated virus serotype 3 (rAAV3), recombinant adenoassociated virus serotype 4 (rAAV4), recombinant adeno-associated virus serotype 5 (rAAV5), recombinant adeno-associated virus serotype 6 (rAAV6), recombinant adeno-associated virus serotype 7 (rAAV7), recombinant adeno-associated virus serotype 8 (rAAV8), recombinant adeno-associated virus serotype 9 (rAAV), serotype variants, for example for enhanced transduction of Miiller glia cells, such as rAAV6 ShHIO (Klimczak et al [
  • the nucleotide sequence encodes for a CRB2 protein comprising an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% preferably at least 100% sequence identity with the amino acid sequences of any one of SEQ ID NO: 40 - 63, 65 - 83, more preferably any one of SEQ ID NO: 40 - 42, most preferably SEQ ID NO:40.
  • Such a CRB2 protein preferably has an intracellular domain of 37 amino acid residues or alternatively an intracellular domain plus transmembrane domain of 63 amino acid residues.
  • these domains in particular are considered most relevant for membrane localization and formation of the Crumbs homologue (CRB) protein complex linked to the actin cytoskeleton of the cell, which are believed to be important to rescue the phenotype and non-toxicity.
  • CRB Crumbs homologue
  • the nucleotide sequence encodes for a CRB2 protein consisting of an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% preferably at least 100%) sequence identity with the amino acid sequences of any one of SEQ ID NO: 40 - 63, 65 - 83, more preferably any one of SEQ ID NO: 40 - 42, most preferably SEQ ID NO:40.
  • the nucleotide sequence encodes for a CRB2 protein comprising or consisting of an amino acid sequence as shown in any one of SEQ ID NO: 40 - 63, 65 - 83, more preferably any one of SEQ ID NO: 40 - 42, most preferably SEQ ID NO: 40.
  • the CRB2 protein comprises a contiguous amino acid sequence that is at least 95% identical to the carboxy (C)- terminal region of 37 contiguous amino acids of a sequence as set forth in SEQ ID NO: 40 - 63, 65 - 83, more preferably any one of SEQ ID NO: 40 - 42, even more preferably SEQ ID NO:40.
  • a CRB2 protein comprising or consisting of an amino acid sequence as defined herein is a functional or, alternatively said, active CRB2 protein.
  • electroretinography is performed.
  • an AAV vector preferably AAV2/9 or AAV2/5, wherein the capsid is AAV9 or AAV5 and the ITRs are AAV2, is generated to allow expression of the CRB2 protein comprising or consisting of an amino acid sequence as defined herein operably linked to a CMV promoter.
  • a construct can be made according to the Examples as presented herein.
  • the AAV vector is administered subretinally to the retina of Crb2 cKO mice (Crb2 F F -ChxlOCre) on postnatal day 14.
  • the contralateral eye receives a control AAV vector which comprises GFP instead of the CRB2 protein to be tested.
  • a positive control animal receives a recombinant AAV expressing CRB2 protein according to SEQ ID NO: 40, 41 or 42.
  • a-wave and b-wave electroretinograms are made as described in Tanimoto et al. (Tanimoto N, Sothilingam V, Seeliger MW; Functional phenotyping of mouse models with ERG. Methods Mol Biol. 2013;935:69-78). Briefly, retinas of anesthetized mice are exposed to light flashes at different intensities (on the x-axis the light intensity expressed as log (cd*s/m 2 )).
  • a CRB2 protein comprising or consisting of an amino acid sequence as defined herein is considered to have CRB2 activity (or to be a functional CRB2 protein) if the maximal b-wave and/or a-wave amplitude (in microvolts) in the electroretinogram is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 fold as compared to the AAV-GFP treated contralateral retina. More preferably, the AAV vector is administered not on postnatal day 14, but on postnatal day 3 or 4, since this may significantly increase the efficacy of treatment.
  • the nucleotide sequence encoding CRB2, modified CRB1 or modified CRB3 is operably linked to expression control elements comprising a promoter that produces sufficient expression of CRB2, modified CRB1 or modified CRB3 protein, respectively, to obtain a therapeutic effect
  • the promoter preferably is selected from the group consisting of: CMV promoter, preferably according to SEQ ID NO: 121, CMV promoter, truncated CMV or minimalCMV promoter, truncated human RLBP1 promoter, human photoreceptor specific rhodopsin kinase promoter and human rod photoreceptor specific rhodopsin promoter.
  • nucleic acid sequence of an illustrative human GRK1 specific promoter (Khani et al, 2007; Boye et al, 2012) which is preferred for use in the present invention is shown in SEQ ID NO: 123.
  • the nucleic acid sequence of an illustrative human RHO specific promoter (Pellissier LP, Hoek RM, Vos RM, Aartsen WM, Klimczak RR, Hoyng SA, Flannery JG, Wijnholds J (2014) Specific tools for targeting and expression of Muller glia cells.
  • Mol Ther Methods&Clinical Dev7: 14009 which is preferred for use in the present invention is shown in SEQ ID NO: 124.
  • the nucleic acid sequence of an illustrative truncated CMV promoter (Pellissier LP, Hoek RM, Vos RM, Aartsen WM, Klimczak RR, Hoyng SA, Flannery JG, Wijnholds J (2014) Specific tools for targeting and expression of Muller glia cells. Mol Ther Methods&Clinical Dev7: 14009) is shown in SEQ ID NO: 133.
  • Particularly preferred gene therapy constructs of the present invention are the following: AAV-hGRKl-CRB2 (specific for rod and cone photoreceptors); AAV- hRHO-CRB2 (specific for rod but not cone photoreceptors); AAV-CMV-CRB2 (allows expression in rod+cone photoreceptors, Muller glia cells, and retinal pigment epithelium); AAV-CMV-CRB2-miRT (that reduces transcription in retinal pigment epithelium); AAV-truncatedRLBPl-CRB2; AAV-truncatedCMV-CRB2.
  • AAV- hGRKl-CRB2 specific for rod and cone photoreceptors
  • AAV-hRHO-CRB2 specific for rod but not cone photoreceptors
  • AAV-CMV-CRB2 are most preferred.
  • the nucleic acid sequence of illustrative micro RNA target sites (miRT's) to lower the expression of AAV transcript containing the miRT sequence in retinal pigment epithelium cells (Karali et al, 2011) which can be used in combination with the present invention is shown in SEQ ID NOs: 125 - 127. These sequences are the predicted and functional target sites in e.g. the AAV-CMV-CRB2-miRT vector, not the miRNA sequences themselves. miRNAs, that recognize and interfere with the translation of, or degrade, the target CRB2-miRT mRNA transcript, are expressed in the RPE. The skilled person is capable of using such miRTs in the present invention (see for example Karali et al. (2011) PLoS One 6(7):e22166. doi: 10.1371/journal.pone.0022166. Epub 2011 Jul 26).
  • recombinant CRB protein preferably CRB2 is expressed in rod and cone photoreceptor cells, but not in retinal pigment epithelium or Miiller glia cells.
  • CRB2 recombinant CRB protein
  • a preferred gene therapy vector of the invention is hGRKl-hCRB2(In5)-spA using AAV2 ITR and AAV5 capsid proteins.
  • the gene therapy vector comprises a nucleotide sequence encoding a modified CRBl protein. Because of the size of e.g. human CRB l, this nucleotide sequence cannot be placed under control of a CMV promoter, since it would result in a construct that is too large for generation of viable AAV virions. Thus, for generation of AAV virions a promoter and enhancer elements must be used that result in a vector of about 4.9 kb at maximum. In such a case, for example the truncated CMV promoter or human photoreceptor specific rhodopsin kinase promoter can be applied.
  • the gene therapy vector according to the invention further comprises operably linked to the nucleotide sequence encoding the CRB protein one or more of the following: inverted terminal repeats such as for example of any wild-type or mutant AAV; a promoter/enhancer such as for example the CMV promoter/enhancer; a wild-type or synthetic transcription splice donor/acceptor site such as for example In5; a wild-type or synthetic transcription poly-adenylation site as for example spA; one or more micro RNA target sites to reduce transcriptional activity in retinal cell types such as for example the retinal pigment epithelium
  • the gene therapy vector according to the invention comprises a wild-type, mutant or codon-optimized DNA sequence encoding wild-type or mutant Crumbs homologue (CRB) proteins of any species.
  • CRB Crumbs homologue
  • a wild-type or synthetic transcription splice donor/acceptor site such as for example synthetic intron (In5) has been inserted in the gene therapy vector for stable transcript processing of CRB2, modified CRBl or modified CRB3.
  • a preferred nucleic acid sequence of an illustrative synthetic intron (In5) in the coding sequence of the Crumbs homologue (CRB) gene is shown in SEQ ID NO: 128.
  • the intron is preferably inserted into CRB2, modified CRBl or modified CRB3 cDNA between two adjacent exons with a sequence of exon NNNAG/intron/GNNN exon, where G, A, T, C stands for one of the four nucleotides, and N stands for any of the four nucleotides.
  • the gene therapy vector according to the invention comprises a nucleotide sequence encoding a CRB2 protein comprising an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%) preferably at least 100%> sequence identity with the amino acid sequence of SEQ ID NO:40 and wherein the promoter is the CMV promoter according to SEQ ID NO: 121.
  • the gene therapy vector according to the invention comprises a nucleotide sequence encoding a CRB2 protein comprising an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%), 97%), 98%), 99%) preferably at least 100%> sequence identity with the amino acid sequence of SEQ ID NO:40 and wherein the promoter is the human photoreceptor specific rhodopsin kinase promoter according to SEQ ID NO: 123.
  • the mutation in the C-terminal part of the amino acid sequence of the modified CRBl or modified CRB3 protein is selected from the group consisting of: i) the PDZ binding domain of CRB l or CRB3 is replaced by amino acid residues 1282 - 1285 of SEQ ID NO:40; ii) the FERM binding domain of CRB l or CRB 3 is replaced by amino acid residues 1251 - 1264 of SEQ ID NO:40; iii) the transmembrane domain of CRB l or CRB 3 is replaced by amino acid residues 1225 - 1247 of SEQ ID NO:40; iv) the 16 C-terminal amino acid residues of CRB l or CRB3 are replaced by amino acid residues 1270 - 1285 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1270 - 1285 of SEQ ID NO:40; v) an amino acid sequence of CRBl or CRB 3 consisting of the FERM binding
  • the C-terminal part of the modified CRB1 or the modified CRB 3 protein are the last 70, 60, 40, 35, 30, 25, 20, 15, 10, 5 residues of the protein.
  • the last 70, 60, 40, 35, 30, 25, 20, 15, 10, 5 residues of the protein that correspond to SEQ ID NO: 1 - 39, 64 (modified CRB1) or SEQ ID NO: 84 - 120 (modified CRB3).
  • the C-terminus of CRB proteins comprises two protein interaction domains: the FERM domain located immediately adjacent to the transmembrane domain and the PDZ domain at the C-terminus. In addition, there are potential phosphorylation sites.
  • Crumbs homologue (CRB) consensus regions as illustrated by reference to amino acid positions of consensus sequence SEQ ID NO:77: Amino acid positions: 265-1515, 1555-2068, and 2083-2146.
  • CRB variable regions Amino acid positions: 1-264, 1516- 1554, and 2069-2082.
  • Other notable regions of the Crumbs homologue (CRB) consensus alignment may or may not include:
  • the present invention relates to a nucleic acid construct, preferably an isolated nucleic acid construct, comprising: a) a nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein and at least one parvoviral inverted terminal repeat (ITR) sequence, wherein preferably the nucleotide sequence encoding a Crumbs homologue-2 (CRB2) protein is operably linked to expression control elements comprising a promoter that is capable of sufficient CRB2 protein expression to obtain a therapeutic effect; and/or b) a nucleotide sequence encoding a Crumbs homologue- 1 (modified CRBl) protein or a Crumbs homologue-3 (modified CRB3) protein, and at least one ITR sequence, wherein preferably the nucleotide sequence encoding a modified CRBl protein or a modified CRB3 protein is operably linked to expression control elements comprising a promoter that is capable of sufficient modified CRBl or modified CRB3 protein expression to
  • this mutation provides substantial identity of modified CRBl or modified CRB3 protein with the C-terminal part of a CRB2 protein.
  • the nucleic acid construct comprising the sequence encoding for modified CRBl or modified CRB3 protein is not toxic in an in vitro and/or an in vivo assay.
  • the in vitro assay comprises transfection of human-derived retinal pigment epithelial cells with the nucleotide sequence encoding the modified CRBl or modified CRB3 protein as described above.
  • the in vivo assay comprises transduction of mouse retinas with the nucleotide sequence encoding the modified CRB 1 or modified CRB3 protein as described above.
  • the nucleic acid construct comprises a nucleotide sequence encoding a CRB2 protein.
  • the mutation in the C-terminal part of the amino acid sequence of the modified CRBl or modified CRB3 protein provides substantial identity of modified CRBl or modified CRB3 protein with the C-terminal part of a CRB2 protein, preferably at least 85, 90, 93, 95, 97, 98, 99 or 100% identity with the C- terminal part of a CRB2 protein, more preferably with the following ranges of amino acid residues of SEQ ID NO:40.
  • the mutation in the C- terminal part of the amino acid sequence of the modified CRBl or modified CRB3 protein is selected from the group consisting of: i) the PDZ binding domain of CRBl or CRB 3 is replaced by amino acid residues 1282 - 1285 of SEQ ID NO:40; ii) the FERM binding domain of CRB l or CRB3 is replaced by amino acid residues 1251 - 1264 of SEQ ID NO:40; iii) the transmembrane domain of CRB l or CRB3 is replaced by amino acid residues 1225 - 1247 of SEQ ID NO: 40; iv) the 16 C-terminal amino acid residues of CRB l or CRB3 are replaced by amino acid residues 1270 - 1285 of SEQ ID NO:40 or by conserved amino acid substitutions of amino acid residues 1270 - 1285 of SEQ ID NO:40; v) an amino acid sequence of CRB l or CRB3 consisting of the FERM binding domain
  • the present invention relates to a virion, comprising a nucleic acid construct according to the present invention, wherein preferably the virion is an AAV virion.
  • the present invention relates to a host cell comprising a nucleic acid construct according to the invention.
  • the host cell is a mammalian or an insect host cell as defined herein above. If the host cell is a mammalian host cell, then preferably a human host cell.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a gene therapy vector according to the invention, a nucleic acid construct according to the invention, or a virion according to the invention and a pharmaceutically acceptable excipient.
  • Pharmaceutically acceptable excipients are well-known by the person skilled in the art. Examples of pharmaceutically acceptable excipients are a buffer, a carrier, a vehicle or a diluent.
  • the pharmaceutical composition further comprises one or more of the following: a lipid, a liposome, a lipid complex, an ethosome, a niosome, a nanoparticle, a microparticle, a liposphere, a nanocapsule, or any combination thereof.
  • the pharmaceutical composition is formulated for administration to the human eye.
  • the composition is administered by direct injection into the retina or the surrounding tissue.
  • the composition needs to be suitable for subretinal or intravitreal injection and thus needs to be a sterile and isotonic fluid, using NaCl or sugars.
  • the invention in a sixth aspect, relates to a kit comprising: (a) a gene therapy vector in accordance with the present invention, a nucleic acid construct according to the present invention, a virion according to the present invention, or a pharmaceutical composition according to the present invention; and (b) instructions for using the gene therapy vector or pharmaceutical composition according to (a) in the prevention, treatment, or amelioration of one or more symptoms of a retinal disorder due to mutations in CRB1 gene.
  • Table 1 List of SEQ ID NO' s with species, genes and accession numbers
  • AAV2 inverted terminal repeat flanked at the 3' end with a Bglll restriction site (AGATCT)
  • AAV2 inverted terminal repeat flanked at the 5' end with a Bglll restriction site (AGATCT)
  • Figure 1 A representative presentation of the localization of CRB 1 in the human retina.
  • Figure 2. A representative presentation of the localization of CRBl in the mouse retina.
  • Figure 3. A representation of the mammalian Crumbs homologue protein family.
  • FIG. 1 Degeneration in the ventral but not in the dorsal retina of CrbF ' Crb2 F/+ ChxlOCre /+ mice.
  • A-E Technovit sections. Left panels, dorsal (superior) retina. Right panels, ventral (inferior) retina.
  • FIG. 5 Electroretinogram b-waves of CrbF / -Crb2 F/+ C x ⁇ 0Cre/+ mice (on 50% C57BL/6J and 50% 129/Ola genetic background) showing loss of retinal activity at 3 months of age. Note that wild-type, CrbF ' and CrbF ' Crb2 F/F mice (not containing ChxlOCre) do not show differences in retinal activity (data not shown). Panel a, scotopic ERG showing loss of rod photoreceptor activity in CrbF ' Crb2 F/+ ChxlOCre retinas (light grey; lower line) vs. CrbF ' Crb2 F/F retinas (dark grey; upper line).
  • Panel b photopic ERG showing loss of cone photoreceptor activity in CrbF ' Crb2 F/+ ChxlOCre retinas (light grey; lower line)) vs. CrbF ' Crb2 F/F retinas (dark grey; upper line).
  • Figure 6 Loss of a separate photoreceptor layer in CrbF ' Crb2 F/F C xlOCre /+ retinas.
  • A-E Technovit sections. Left panels, control retina. Middle panels, Crbl +/ ⁇ Crb2 F/F C xlOCre /+ retinas. Right panels, CrbF ' Crb2 F/F C xlOCre /+ retinas showing absence of a separate photoreceptor layer and mislocalized retinal cells.
  • Figure 7. Loss of retinal activity in CrblCrb2 cKO compared to Crbl and Crb2 cKO retinas. Panels a and c, measured ERGs at 3 months of age.
  • Panels b and d measured ERGs at 1 months of age. Panels a and b, scotopic. Panels c and d, photopic. (Note, at 3 months of age, the very good separation of confidence intervals in b-wave amplitude between Crbl KO and Crb2 cKO retinas.)
  • the lines in the figures represent the following: the upper most line concerns Crbl KO, second from the top is Crb2 KO, third from the top is Crbl +/' Crb2 F/F ChxlOCre (heterozygote Crbl +/ ⁇ homozygote floxed Crb2 F/F heterozygote ChxlOCre), the bottom line is Crb ⁇ ' Crb2 F/F ChxlOCre (homozygote Crbl ' ' homozygote floxed Crb2 F/F heterozygote ChxlOCre).
  • FIG. 8 Upon subretinal injection, AAV9-CMV-GFP and ShHIOY-CMV-GFP infect Miiller glia cells and photoreceptors.
  • GCL ganglion cells
  • PRC photoreceptor cells
  • RPE retinal pigment epithelium cells.
  • Figure 9 Expression of short CRBl (SEQ ID NO. 3) in Crbl KO retinas using subretinal injection of AAV2/9-CMV-sCRB 1 vectors.
  • Panel a control Crbl KO retina.
  • Panel b Crbl KO retina expressing sCRBl upon transduction with AAV2/9-CMV- sCRBl viral particles.
  • FIG. 10 Representative experiment showing rescue of loss of retinal activity.
  • Crb2 cKO retinas were injected at postnatal day 23 subretinally with 10 10 AAV2/ShH10Y- CMV-CRB2 or AAV2/ShH10Y-CMV-GFP viral particles and analyzed for ERG and immunohistochemistry at 3 months of age.
  • Panel a electroretinogram scotopic b-wave showing rescue of retinal activity in the right Crb2 cKO eye transduced with AAV2/ShH10Y-CMV-CRB2 (dark line), compared to the left eye of the same Crb2 cKO transduced with AAV2/ShH10Y-CMV-GFP (faint line).
  • FIG. 11 Specific transduction of Miiller glia cells using 10 10 AAV2/6-RLBP 1 -GFP viral particles containing the human RLBPl promoter (SEQ ID NO. 122) upon intravitreal injection (specific infection of Miiller glia cells). Retinas were collected 3 weeks post-infection.
  • Panel a scanning-laser-ophtalmoscopy (SLO).
  • Panel b SLO showing fluorescent cells.
  • Panel c immunohistochemistry showing specific expression of GFP in Miiller glia cells.
  • FIG. 12 AAV6 and ShHIOY capsids transduce adult human Miiller glia cells.
  • One ⁇ 10 13 genome copies per ml of AAV2/6-CMV-GFP-WPRE-pA (panel a) or AAV6 variant AAV2/ShH10-CMV-GFP-WPRE-pA (panel b) was applied to pieces of cultured adult human retina. GFP expression was detected in Miiller glia cells.
  • FIG. 13 GFP and CRBl protein expression in cell lines.
  • Western Blotting of HEK293T cell lysates transfected with the calcium phosphate method and 10 ⁇ g of pAAV-CMV-GFP-WPRE-pA or pAAV-CMV-hCRBl-pA vectors showed subsequent CRBl and GFP protein levels.
  • RPE-derived ARPE-19 cells expressed normal amount of GFP, CRBl protein is just above detection level in three times overloaded protein lysates.
  • Figure 14 Rescue of loss of retinal function by subretinal injection of AAY2/9-CRB2 viral particles in Crb mutant mouse eyes; failure of rescue of retinal function by AAV2I9-CRB1 viral particles.
  • AAV2/9-CMV -GFP a- c, g-h
  • AAV2-minimalCMV-CR57- spA minimal CMV presented as SEQ ID NO: 133 in the Sequence listing
  • AAV2/9-minCMV-Gi P d-f
  • Scotopic b- wave amplitudes (a, d, g) and a-wave amplitudes (b, e, h), and photopic b-wave amplitudes (c, f) are indicated.
  • CRB2 vectors rescued loss of retinal function in two different Crb mutant mouse models (a-c, g-h), whereas CRBl vectors did not rescue loss of retinal function (d-f).
  • FIG. 15 Toxicity of CRB proteins tested by intravitreal injection of AAV2/ShH10Y- or AAV2/ShH10Y-CMV-CR52-/w5-3 ⁇ 43 ⁇ 44 viral particles in Crb mutant mouse eyes, i.e. CRB2 or CRBl DNA operably linked to the promoter that is indicated, flanked by AAV2 ITRs and packaged in ShHIOY capsid proteins,.
  • the eyes were analyzed at 3 months of age by electroretinography under scotopic (dark-adapted; a-b, c-d) or photopic (light- adapted; e) conditions. Scotopic b-wave amplitudes (a, c) and a-wave amplitudes (b, d), and photopic b-wave amplitudes (e) are indicated. No statistical significant differences in retinal function were detected for intravitreally applied CRB2 vectors compared to GFP control vectors (a-b). Intravitreally applied CRBl vectors showed strongly reduced retinal responses upon expression of CRBl vectors, suggesting toxic effects by CRBl vectors.
  • Retinas of Cr67 ⁇ /+ Cr62 / ChxlOCre/+ mice show to some extent a similar but more severe phenotype than observed in Cr b2 F/F C x 10 Cre/+ retinas. Electroretinography showed a significant loss of retinal activity at 1 month of age that progressed quickly. The phenotype starts already at E15.5 (at this time point similar to E17.5 in Cr b2 F/F C x 10 Cre/+ retinas), with disruptions at the outer limiting membrane, and rosettes of retinal cells can be detected.
  • a major difference of these mouse retinas compared to the Cr£2 / ChxlOCre/+ retinas is the aberrant localization of several retinal cell types. E.g., some amacrine cells ectopically localize in the photoreceptor layer, and some cone and rod photoreceptors ectopically localize at the ganglion cell layer. Nevertheless, in these retinas there is still three nuclear layers (outer and inner, and ganglion) and two plexiform layers (outer and inner) suggesting that the lamination of the retina is grossly normal.
  • Retinas of Crbl ⁇ Crb ⁇ Chx 10Cre/+ mice show the most severe phenotype (Figure 6). Electroretinography showed a severe loss of vision at 1 month of age (though there is still some retinal activity; Figure 7). These retinas do not show a separate photoreceptor layer (no outer and inner segment, nuclear or outer plexiform layer) and no outer plexiform layer but a single broad nuclear layer, an inner plexiform layer, and a ganglion cell layer.
  • the nuclear layer contains nuclei of rod and cone photoreceptors, bipolar, horizontal, amacrine and Miiller glia cells, but surprisingly also nuclei of ganglion cells.
  • the inner plexiform layer only occasionally contains cell nuclei.
  • the ganglion cell layer that normally contains nuclei of ganglion and displaced amacrine cells contains in addition nuclei of rod photoreceptors, bipolar, horizontal, Miiller glia cells. So, whereas there is a laminated retina, several early as well as late born cells localized ectopically. Furthermore, there was a significant increase in dividing retinal progenitor cells at E15.5, E17.5, PI and P5.
  • Crbl / -Crb2 F/+ ChxlOCre/+ mice on 99.9% C57BL/6J background are being produced and will provide less inter-mouse variation.
  • Cr 2 / ChxlOCre/+ retinas which mimic loss of CRBl in retinitis pigmentosa patients, as the best for rescue studies since their electroretinograms are easier to interpret.
  • transcripts of the human CRB 1 gene there are alternative transcripts of the human CRB 1 gene.
  • the sequence of CRB2 (SEQ ID NO:40) is very similar to the sequence of this naturally occurring short variant of CRB l (sCRBl or SCRB1AE3/4).
  • AAV vectors with the CMV promoter, the short CRB l, a synthetic intron (In5) in the short CRBl cDNA sequence, and a synthetic spA.
  • AAV9 AAV serotype 9
  • results were obtained with vector packaged in AAV serotype 5 (AAV5).
  • AAV2/9-CMV-hCRB lAE3/4In5- spA (1.00 x 10 10 delivered vector genomes) plus a ten-fold lower dose of AAV2/9- CMV-GFP-WPRE-pA (1.00 x 10 9 delivered vector genomes) into the left eye of retinas lacking CRBl with reduced levels of CRB2 (Crbl "/" Crb2 flox/+ ChxlOCre retinas).
  • the contralateral control eye received one ⁇ of AAV2/9-CMV-GFP-WPRE-pA (1.00 x 10 10 delivered vector genomes).
  • ChxlOCre retinas show progressive loss of retinal function from 1 to 3 to 6 months of age (data not shown).
  • the treated eyes showed expression of short CRBl in a large region of the retina at the "outer liming membrane" of Miiller glia cells and photoreceptors, and in retinal pigment epithelium.
  • the cause of these toxic effects is to be further analysed and may for example be e.g.
  • an immune-response in the CRB1 naive Crbl knockout retina ectopic expression effect, incompatibility of mouse and human CRB1 protein, differences between short and full length CRB1, interference of short CRB1 with the expression of other CRB1 transcripts or proteins, dose-dependent toxicity, untimely expression of short CRB1), and it might be related to the inability in producing continuous high level expression of short (or full length) CRB 1 in cultured cell lines.
  • Preliminary studies expressing the short CRB1 in wild-type C57BL/6J retina showed toxicity as well, suggesting that the toxicity is not only due to the expression of short human CRB1 in immune-naive Crbl knockout retina.
  • CRB2 expression was well tolerated in cell lines.
  • Expression of CRB2 in Miiller glia cells or photoreceptor cells or retinal pigment epithelium did not result in toxic effects. More specifically, expression of CRB2 in Muller glia cells or photoreceptor cells or retinal pigment epithelium did not result in a detectable loss of the photoreceptor layer and/or the retinal pigment epithelium layer. This lack of toxic effects of CRB2 expression in Muller glia cells and photoreceptor cells and retinal pigment epithelium is relevant to the development of future clinical applications. Note that we used very high levels of AAV-CRB2 vector (10 10 delivered vector genomes) but toxic effects were not detected.
  • the inventors evaluated whether delivery of a species-specific version of Crumbs homologue (CRB) (i.e., human) to Muller glia cells and photoreceptors of the postnatal Crb2 cKO mouse could restore function to these cells.
  • Serotype 6 (variant ShHIOY) AAV vectors were used to deliver human CRB2 subretinally to Muller glia cells and photoreceptors of postnatal day 23 (P23) Crb2 cKO mice.
  • Electroretinogram (ERG) and behavioral testing were used to assess visual function and immunocytochemistry was used to examine therapeutic transgene expression, Crumbs homologue (CRB) complex protein localization and preservation of retinal structure in treated and untreated eyes.
  • This example demonstrates that an AAV vector subretinally delivered to the left eyes of P23 Crb2 cKO mice facilitated expression of wild-type CRB2, restoration of visual function and behavior, and preservation of rod and cone photoreceptors.
  • retinal function (ERG) was analyzed in treated and untreated eyes. In some experiments, ERG was performed every two weeks after 4 weeks until 10 weeks post injection (the latest time point evaluated). At 10 weeks post injection, all animals were sacrificed and their treated and untreated retinas were evaluated for expression of CRB2 and localization of Crumbs homologue (CRB) complex proteins.
  • CRB Crumbs homologue
  • Crb2(flox/flox) mice were generated at the inventor's facilities. ChxlOCre heterozygote embryos were obtained from a living stock at The Jackson Laboratory (Bar Harbor, ME, USA). Heterozygotes were mated at the inventors' facilities to produce Crb2(flox/flox)ChxlOCre homozygous mice and isogenic Crb2(flox/+)ChxlOCre control offspring (both heterozygous for ChxlOCre). All mice were bred and maintained in a centralized facility at the inventors' institution under a 12hr/12hr light/dark cycle. Food and water were available ad libitum.
  • AAV vectors with serotype 6 variant ShHIOY capsid proteins and AAV2 ITR and Rep proteins were used to deliver human CRB2 (hCRB2) as they have been shown to exhibit robust transduction efficiency and a faster onset of expression in retinal Miiller glia cells as well as photoreceptors than other AAV serotypes.
  • the serotype 6 variant ShHIOY AAV capsid was provided by Dr. John Flannery (University of California, Berkeley, CA, USA).
  • AAV serotype 5 was obtained from Plasmid Factory.
  • AAV serotype 9 was obtained from Dr. Joost Verhaagen (Netherlands Institute for Neuroscience).
  • CMV ubiquitous cytomegalovirus
  • SEQ ID NO: 121 The nucleic acid sequence of an illustrative ubiquitous CMV promoter which was used in the studies is shown in SEQ ID NO: 121.
  • the CMV promoter is flanked at the 5' sequence with a Bglll restriction site (AGATCT).
  • a synthetic intron (In5) inserted in the CRB2 cDNA was used for stable transcript processing of CRB2.
  • the nucleic acid sequence of an illustrative synthetic intron (In5) in the coding sequence of the Crumbs homologue (CRB) gene is shown in SEQ ID NO: 128.
  • the intron was inserted into CRB2 cDNA between two adjacent exons with a sequence of exon NNNAG/intron/GNNN exon, where G, A, T, C stands for one of the four nucleotides, and N stands for any of the four nucleotides.
  • a synthetic poly-adenylation (spA) sequence was used for efficient termination of transcription.
  • the nucleic acid sequence of an illustrative synthetic polyadenylation region (Levitt et al, 1989) in between the stop codon behind the translated region of the Crumbs homologue (CRB) gene and the 3 'flanking inverted terminal repeat which was used is shown in SEQ ID NO: 129.
  • the synthetic polyadenylation site is flanked at the 3 'sequence with a Bglll restriction site (AGATCT).
  • AGATCT Bglll restriction site
  • SEQ ID NO: 130 The nucleic acid sequence of an illustrative 5' untranslated region located in between the CMV promoter and the translated region of the Crumbs homologue (CRB) gene which was used is shown in SEQ ID NO: 130.
  • the CMV-hCRB2In5-spA fragment, containing Bglll restriction sites at the 5' and 3' ends, with sequence identified in SEQ ID NO:40 was synthesized by GenScript (Piscataway, NJ, USA).
  • GenScript Procataway, NJ, USA
  • the Bglll CMV-hCRB2In5-spA fragment was cloned into pUC57 (Thermo Fisher Scientific, Waltham, MA, USA) containing two inverted terminal repeats (ITRs) of AAV2 flanked by Bglll restriction sites (SEQ ID NO: 131 and 132).
  • the resulting AAV-hCRB2 plasmid of 4.9 kb contained the sequence identified in SEQ ID NO:40 and was sequence verified.
  • AAV vectors were packaged and purified by iodixanol gradient ultra- centrifugation according to previously published methods (Zolotukhin et al, 1999; Hermens et al, 1999; Ehlert et al, 2010). Viral particles were diluted, washed and concentrated using an Amicon 100 kDa MWCO Ultra- 15 device (Millipore, Billerica, MA, USA) in Dulbecco's Balanced Salt Solution (Life Technologies, Bleiswijk, Netherlands) and titered by quantitative real-time PCR (Aartsen et al, 2010).
  • Resulting titers were 1.00 x 10 13 viral genomes per ml (vg/ml) for AAV2/ShH10Y-CMV-hCRB2 or AAV2/9-CMV-hCRB2 (AAV2 ITR and Rep proteins; AAV9 capsid proteins) or AAV2/5-CMV-hCRB2 (AAV2 ITR and Rep proteins; AAV5 capsid proteins).
  • AAV2/ShH10Y-CMV-hCRB2 (1.00 x 10 10 delivered vector genomes) plus a ten- fold lower dose of AAV2/ShH10Y-CMV-GFP- WPRE-pA (1.00 x 10 9 delivered vector genomes) was delivered subretinally at postnatal day 23 (P23) to the left eye of each Crb2(flox/flox)ChxlOCre mouse.
  • the contralateral control right eye was injected with one ⁇ of AAV2/ShH10Y-CMV-GFP- WPRE-pA (1.00 x 10 10 delivered vector genomes). Subretinal injections were performed as previously described (Aartsen et al, 2010).
  • retinal detachment corresponds to the area of viral transduction (Cideciyan et al, 2008; Timmers et al, 2001).
  • ERGs electroretinograms
  • PC-based control and recording unit Toennies Multiliner Vision; Jaeger/Toennies, Hochberg, Germany
  • Initial ERG measurements were recorded at 4 weeks' postinjection, and each subsequent 2 weeks thereafter, until 10 weeks post-injection (the latest time point evaluated in the study).
  • Age matched isogenic controls were recorded alongside treated animals at every time point.
  • mice were dark-adapted overnight (more than 12 hours) and anesthetized with a mixture of 100 mg/kg ketamine, 20 mg/kg xylazine and saline in a 1 : 1 :5 ratio, respectively.
  • Pupils were dilated with 1% tropicamide and 2.5% phenylephrine hydrochloride.
  • a heated circulating water bath was used to maintain the body temperature at 38°C.
  • Hydroxypropyl methylcellulose 2.5% was applied to each eye to prevent corneal dehydration.
  • Full-field ERGs were recorded using custom, gold wire loop corneal electrodes. Reference and ground electrodes were placed subcutaneously between the eyes and in the tail, respectively.
  • Scotopic rod recordings were elicited with a series of white flashes of seven increasing intensities (0.1 mcds/m 2 to 1.5 cds/m 2 ). Interstimulus intervals for low intensity stimuli were 1.1 second. At the three highest intensities (100 mcds/m 2 , 1 cds/m 2 and 5 cds/m 2 ), interstimulus intervals were 2.5, 5.0 and 20.0 seconds, respectively. Ten responses were recorded and averaged at each intensity. Mice were then light adapted to a 100 cds/m 2 white background for 2 min. Photopic cone responses were elicited with a series of five increasing light intensities (100 mcds/m 2 to 12 cds/m 2 ).
  • B-wave amplitudes were defined as the difference between the a- wave troughs to the positive peaks of each waveform.
  • ERGs recordings were elicited with a series of light pulses of increasing intensities (2.7 cds/m 2 to 25 cds/m 2 , logarithmically spread over 10 levels. Pulse lengths ranged from 0.5 to 5 msec. Between pulses there was a delay of approximately 2 seconds (.5Hz). Thirty responses were recorded and averaged at each intensity. No extra delay was introduced for the transition from one intensity level to the next. Between pulses, no background lighting was present.
  • the a-wave trough was defined as the minimum response between 0 and 30 milliseconds after stimulus onset.
  • the b-wave peak was defined as the maximum response between 15 and 100 milliseconds after stimulus onset.
  • the a-wave amplitude was defined as the difference between the baseline and the a-wave trough, whereas the b-wave amplitude was defined as the difference between the b-wave peak and the a-wave trough.
  • mice Ten weeks post-injection, P23-treated Crb2 cKO mice and age matched isogenic controls were dark adapted for 2 hr. Immediately following dark adaptation, mice were sacrificed under dim red light (>650 nm). The limbus of injected and un-injected eyes was marked with a hot needle at the 12:00 position, facilitating orientation. Enucleation was performed under dim red light and eyes were placed immediately in 4% paraformaldehyde. Eyes that were to be used for cryosectioning were prepared according to previously described methods (Haire et al, 2006). Briefly, corneas were removed from each eye, leaving the lens inside the remaining eye cup. A small "V" shaped cut was made into the sclera adjacent to the burned limbus to maintain orientation.
  • Retinal cryosections and whole mounts were washed 3 x in IX PBS. Following these washes, samples were incubated in 0.5% Triton X-100® for 1 hr in the dark at room temperature. Next, samples were blocked in a solution of 1 % bovine serum albumin (BSA) in PBS for 1 hr at room temperature. Retinal sections were incubated overnight at 37°C with a rabbit polyclonal CRB2 antibody EP 13 or SKI 1 (1 : 1000 and 1 :200, respectively; provided by Dr. Penny Rashbass, University of Sheffield, UK) diluted in 0.3% Triton X-100®/1 % BSA. Following primary incubation, retinal sections and whole mounts were washed 3X with IX PBS.
  • BSA bovine serum albumin
  • Retinal sections were incubated for 1 hr at room temperature with IgG secondary antibodies tagged with Cyanine dye Cy5 (Molecular Probes, Eugene, OR, USA) diluted 1 :500 in lx PBS. Following incubation with secondary antibodies, sections and whole mounts were washed with lx PBS. Retinal sections were counterstained with 4', 6'- diamino-2-phenylindole (DAPI) for 5 min at room temperature. After a final rinse with IX PBS and water, sections were mounted in an aqueous-based medium (DAKO) and cover-slipped. Retinal whole mounts were oriented on slides with the superior (dorsal) portion of the retina positioned at the 12:00 position. Samples were mounted in DAKO and cover-slipped.
  • DAPI aqueous-based medium
  • Retinal sections were analyzed with confocal microscopy (Leica TCS SP5 AOBS Spectral Confocal Microscope equipped with LCS Version 2.61, Build 1537 software, (Bannockburn, IL, USA). All images were taken with identical exposure settings at either 20x or 63x magnification. Excitation wavelengths used for DAPI and CRB2 stains were 405 nm and 650 nm, respectively. Emission spectra were 440-470 nm and 670 nm, respectively.
  • Retinal whole mounts were analyzed with a widefield fluorescent microscope (Axioplan 2) (Zeiss, Thornwood, NY, USA) equipped with a Qlmaging Retiga 4000R Camera and Qlmaging QCapture Pro software (Qlmaging, Inc., Surrey, BC, Canada). Quadrants of each whole mount were imaged at 5x under identical exposure settings and then merged together in Photoshop® (Version 7.0) (Adobe, San Jose, CA, USA).
  • CRB 1 -deficiency affects both Miiller glia and photoreceptors in LCA8 and RP patients due to mutations in the CRBl gene.
  • the ubiquitous CMV promoter was therefore chosen for this study as a means of targeting both cell types.
  • the AAV6 variant ShHIOY capsid was chosen because it infects upon subretinal injection efficiently in vivo mouse Miiller glia and photoreceptors (and infects e.g. in vitro human retinal Miiller glia cells, see Fig. 12).
  • hCRB2 expression was occasionally found in the retinal pigment epithelium.
  • the retinal pigment epithelium also expresses Crumbs homologue (CRB) complex members such as PALSl (Park et al, 2011), albeit at lower levels than at the outer limiting membrane (Pellissier LP, Lundvig DM, Tanimoto N, Klooster J, Vos RM, Richard F, Sothilingam V, Garcia Garrido M, Le Bivic A, Seeliger MW, Wijnholds J. Hum Mol Genet. 2014 Jul 15;23(14):3759-71).
  • CB Crumbs homologue
  • hCRB2 Overexpression of hCRB2 in the wild-type RPE cells in the Crb2 cKO did not result in noticeable altered morphology or function of retinal pigment epithelium. Notably however, the CMV promoter construct did not drive therapeutic hCRB2 expression outside the photoreceptor cells, Miiller glia cells and retinal pigment epithelium. This lack of off target expression is relevant to the development of future clinical applications. If required, overexpression in retinal pigment epithelium can be decreased by the use of micro-RNA target sites (miRT's) specific for miRNAs expressed in retinal pigment epithelium cells (Karali et al., 2011).
  • miRT's micro-RNA target sites
  • CRB 1 -deficiency in humans causes LCA8 and progressive RP very well detectable by ERG
  • CRB 1 -deficiency in mice causes late- onset retinal degeneration and degeneration limited to one quadrant of the retina and not detectable by ERG.
  • Our immuno-electron microscopy data showed that in mice CRB1 is restricted to the "outer limiting membrane" of Miiller glia cells, whereas in humans CRB1 is localized to the "outer limiting membrane" of Miiller glia cells and photoreceptors.
  • mice CRB2 is localized to the "outer limiting membrane" of Miiller glia cells and photoreceptors, whereas in humans CRB2 is restricted to the "outer limiting membrane” of Miiller glia cells.
  • Our analysis of mice lacking CRB1, mice lacking CRB2, mice lacking CRB 1 with reduced levels of CRB2, mice lacking CRB2 with reduced levels of CRB1, and mice lacking both CRB1 and CRB2 suggest very similar functions for CRB1 and CRB2.
  • the functions of Crumbs homologue (CRB) proteins are exchangeable e.g.
  • the human CRB1 protein can rescue partially the phenotype in fruit flies lacking Crumbs (Crb) protein (den Hollander et al., 2001), and the zebrafish CRB2B protein can rescue the phenotype in zebrafish lacking CRB2A protein (Omori & Malicki, 2006).
  • mice Short human CRBl was overexpressed in retinas lacking CRBl protein expression and with reduced levels of CRB2 protein. Thus, these mice still have functional native CRB2 protein in Miiller glia cells and photoreceptor cells since CRB2 in mouse retina is present in both cell types. It is conceivable that this remaining mouse CRB2 protein is capable of taking over the function of the CRBl protein.
  • mice on 50% C57BL/6J and 50% 129/Ola genetic background were less suitable to test rescuing of the phenotype in the retina. Control mice and mutant mice are significantly different in retina activity as measured using electroretinography. However, there is quite some variation in experimental animals and as a consequence the confidence intervals are close to one another.
  • mice As far as rescuing the phenotype is concerned, the mouse model is still suboptimal and could be further optimized by backcrossing to 99.9% C57BL/6J. Recently, trials were initiated in mice (on 75% C57BL/6J and 25% 129/Ola genetic background) lacking CRBl and having reduced levels of CRB2 using human CRB2 in AAV9 conform to the experimental setting as outlined above.
  • ERG rescue results were obtained using AAV2/9-CMV-CRB2 (1.00 x 10 10 delivered vector genomes) viral particles subretinally injected into P14 Crbl " Crb2 ChxlOCre retinas (on 75% C57BL/6J and 25% 129/Ola genetic background) that were analyzed at 4 months of age.
  • Retinas of CrblCrb2 /+ conditional knock-out mice on 75% C57BL/6J and 25% 129/Ola genetic background show a big difference in retina activity at 3 months of age.
  • the retinas of CrblCrb2 /+ mutant mice are rescued phenotypically, and the confidence intervals are separated and well interpretable.
  • the present Example indicates that the phenotype, measured as retina activity using electroretinography, in the eyes that show expression of recombinant human CRB2 is rescued. In absence of expression of recombinant human CRB2 the phenotype is not rescued.
  • CRB proteins can be tested using human-derived retinal pigment epithelial cells according to the following Example.
  • ARPE19 cells (ATCC CRL-2302) are transfected with one of the different (modified) CRB constructs (e.g. CRB 1, sCRBl, CRB 2 isoform 1, CRB2 isoform 2, CRB2 isoform 3, CRB 3 etc.) together with a control GFP construct (Aartsen et al. (2010) PLoS One 5:el2387;GFAP-driven transgene expression in activated Miiller glial cells following intravitreal injection of AAV2/6 vectors; UniProtKB/Swiss-Prot sequence P42212) using the calcium phosphate method (described e.g.
  • CRB2 construct As a control a CRB2 construct is used (CRB2 sequence: SEQ ID NO:40). The CRB constructs are used in equimolar amounts and a total amount of 20 ⁇ g of DNA is added per petridish. CRB constructs are made as described in Example 2.1. Briefly, CRB constructs are made by chemical synthesis and subcloned into pUC57. These constructs comprise AAV2 ITRs (SEQ ID NO: 131 and 132), CMV promoter (SEQ ID NO: 121), CRB cDNA to be tested (e.g. SEQ ID NO:40 or other CRB sequence, Intron 5 (SEQ ID NO: 128), and synthetic pA (SEQ ID NO: 130).
  • AAV2 ITRs SEQ ID NO: 131 and 132
  • CMV promoter SEQ ID NO: 121
  • CRB cDNA to be tested e.g. SEQ ID NO:40 or other CRB sequence, Intron 5 (SEQ ID NO: 128),
  • the GFP construct is used as internal transfection control in a fixed amount. For example, 18 ⁇ g of CRB construct plus 2 ⁇ g of GFP construct is used. In this way, a series of equimolar plasmid concentrations can be tested while adding the same amount of DNA, such as for example 2, 4, 8 or 16 ⁇ g of CRB construct, plus 18, 16, 12 or 4 ⁇ g of GFP construct, respectively.
  • ARPE19 cells are plated in duplicate at 30% of confluence in a 10 cm petridish in DMEM supplemented with 10% Fetal Bovine Serine and penicillin/streptomycin. After refreshing the medium 2 hours before transfection, the transfection mix is prepared with 20 ⁇ g of DNA in 500 ⁇ of 0,25M CaCl 2 and TE (10 mM Tris, 1 mM EDTA pH 8) buffer per dish. While constantly vortexing, 500 ⁇ of 2x HBS (281 mM NaCl, 100 mM Hepes, 1.5 mM Na 2 HP0 4 , pH 7.12) are added drop wise to the transfection mixture and the complete mix is directly added to the cells for overnight incubation.
  • 2x HBS 281 mM NaCl, 100 mM Hepes, 1.5 mM Na 2 HP0 4 , pH 7.12
  • the medium is refreshed in the following morning. Two days later (i.e. 72 h after transfection), the attached and floating cells are harvested separately (one duplicate) and together (the second duplicate) and after centrifugation, resuspended in 1 mL of Phosphate Buffer Saline (137 mM NaCl, 2.7 mM KC1, 10 mM Na 2 HP0 4 and 1.76 mM KH 2 P0 4 ). Subsequently, cells are tested for:
  • Trypan Blue staining was performed using the Standard protocol by Life Technologies as outlined below.
  • Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the hemacytometer. If cells take up trypan blue, they are considered non- viable.
  • % viable cells [1.00 - (Number of blue cells ⁇ Number of total cells)] ⁇ 100
  • EXAMPLE 4 GENE REPLACEMENT THERAPY IN Crb l(-/- )Crb2(flox/+)ChxlOCre and Crb2(flox/flox)ChxlOCre MICE USING AAV2/9-CMV- CRB2-In5
  • the retinal pigment epithelium e.g. the Crb l "/" Crb2 flox/+ ChxlOCre on 75% C57BL/6J and 25% 129/Ola genetic background
  • Crb2 cKO mice 99.9% C57BL/6J background
  • AAV-mediated transfer of CRB2 using AAV2/9-CMV-hCRB2-In5 to CrblCrb2 flox/+ cKO retina restored vision to these animals as evidenced by ERG.
  • Subretinal AAV-mediated transfer of CRB2 using 1 ⁇ . of 2.10 10 genome copies of AAV9 viral particles containing 4.9 kb AAV2-CMV-hCRB2-In5 to CrblCrb2 flox/+ cKO retina or Crb2 cKO retina restored vision to these animals as evidenced by ERG, Figure 14 (a-c, g-h).
  • EXAMPLE 5 LACK OF GENE REPLACEMENT THERAPY IN Crbl(-/- )Crb2(flox/+)ChxlOCre USING AAV2/9-CMV-CRB 1
  • Toxicity of CRB proteins can be tested using Crbl(-/-)Crb2(flox/+)ChxlOCre mice according to the following Example.
  • Crbl(-/-)Crb2(flox/+)ChxlOCre mouse retinas are intravitreally injected with a (modified) CRB construct (e.g. CRBl, short CRB l, CRB2 isoform 1, CRB2 isoform 2, CRB2 isoform 3, CRB3 etc.) in a recombinant AAV expression vector in one eye, whereas the contralateral eye receives a control A V-GFP construct (Aartsen et al. (2010) PLoS One 5:el2387;GFAP-driven transgene expression in activated Miiller glial cells following intravitreal injection of AAV2/6 vectors; UniProtKB/Swiss-Prot sequence P42212).
  • a (modified) CRB construct e.g. CRBl, short CRB l, CRB2 isoform 1, CRB2 isoform 2, CRB2 isoform 3, CRB3 etc.
  • the eyes are treated with the vectors using the AAV transduction method (described e.g. in Aartsen et al. (2010) PLoS One 5:el2387;GFAP-driven transgene expression in activated Miiller glial cells following intravitreal injection of AAV2/6 vectors).
  • Control animals receive a AAV-CRB2 construct in one eye and the control AAV-GFP construct in the contralateral eye (CRB2 sequence: SEQ ID NO:40).
  • the AAV-CRB constructs are intravitreally injected into the eyes of Crbl(-/- )Crb2(flox/+)ChxlOCre mice in equimolar amounts and a total amount of 1 ⁇ L ⁇ of 5.10 9 to 10 10 genome copies of AAV2/ShH10Y-(CMV or minimalCMV)-CRB and in the contralateral control eye with the same amount of AAV2/ShH10Y-(CMV or minimalCMV)-GFP.
  • AAV-CRB constructs are made as described in Example 2.1. Briefly, CRB constructs are made by chemical synthesis and subcloned into pUC57.
  • constructs comprise AAV2 ITRs (SEQ ID NO: 131 and 132), CMV promoter (SEQ ID NO: 121) or minimal CMV promoter, CRB cDNA to be tested (e.g. SEQ ID NO:40 or other CRB sequence, synthetic pA (SEQ ID NO: 130) and an optional Intron 5 (SEQ ID NO: 128).
  • the GFP construct is used as internal transduction control in a fixed amount. Plasmids are packaged in AAV serotype ShHIOY capsids.
  • CrblCrb2 F/+ cKO Three to seven Crbr A Crb2 F/+ ChxlOCre Tg/+ (CrblCrb2 F/+ cKO) are injected at 2 weeks of age intravitreally with 1 iL of 5.10 9 to 10 10 genome copies of CRB or control GFP viral particles.
  • In vivo retinal function is to be analyzed at 3 to 5 months of age by electroretinography under scotopic (dark-adapted overnight) or photopic (light-adapted with a background illumination of 30 cd/m2 starting 10 minutes before recording) conditions.
  • Mice are anaesthetized using ketamine (66.7 mg/kg body weight) and xylazine (1 1.7 mg/kg body weight).
  • the pupils are dilated and single royal blue-flash stimuli range from -3 to 1.5 log cd s/m2. Twenty responses are averaged with inter- stimulus intervals of 2 s. A-wave responses revealed direct photoreceptor functions (rods and cones under scotopic and only from cones under photopic conditions) and B- waves revealed the retinal activities. A representative experiment is shown in Figure 15.
  • CRB proteins Retinal expression of CRB proteins upon intravitreal transduction in CrblCrb2 F/+ cKO or Crb2 cKO eyes is examined by standard immunohistochemistry using antibodies against the respective CRB proteins (e.g. anti-CRB2 or anti-CRB l or anti- CRB3 as in van de Pavert et al., J. Cell Science, 2004).
  • antibodies against the respective CRB proteins e.g. anti-CRB2 or anti-CRB l or anti- CRB3 as in van de Pavert et al., J. Cell Science, 2004.
  • Cideciyan A. V. (2010). Prog Retin Eye Res 29, 398-427.
  • Cideciyan A. V. (2013). Proc Natl Acad Sci USA 110, E517-E525.
  • CRB2 acts as a modifying factor of CRBl -related retinal dystrophies in mice.

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Abstract

La présente invention a trait à un composé thérapeutique homologue de Crumbs (CRB) destiné à être utilisé en tant que médicament ou dans le cadre d'un procédé de traitement ou de prévention, par exemple pour le traitement ou la prévention d'un trouble rétinien dû à des mutations du gène Crumbs homologue-1 (CRB1), tel que l'amaurose congénitale de Leber 8 (LCA8) ou la rétinite pigmentaire 12 (RP12). L'invention concerne en particulier un vecteur viral recombinant comprenant CRB2 ou des formes non toxiques modifiées de CRB1 ou CRB3 qui ressemblent à CRB2.
PCT/NL2014/050549 2013-08-05 2014-08-05 Composition d'homologues de crumbs et vaa recombinants et procédés pour traiter la lca-8 et la rp progressive WO2015020522A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US14/910,302 US11246947B2 (en) 2013-08-05 2014-08-05 Recombinant AAV-crumbs homologue composition and methods for treating LCA-8 and progressive RP
CA2919995A CA2919995A1 (fr) 2013-08-05 2014-08-05 Composition d'homologues de crumbs et vaa recombinants et procedes pour traiter la lca-8 et la rp progressive
AU2014305218A AU2014305218B2 (en) 2013-08-05 2014-08-05 Recombinant AAV-Crumbs homologue composition and methods for treating LCA-8 and progressive RP
EP14761426.7A EP3030665B9 (fr) 2013-08-05 2014-08-05 Composition homologue de miettes aav recombinante et procédés pour traiter le lca-8 rp et rp progressif
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